Clove essential oil-in-cyclodextrin-in-liposomes in the aqueous and lyophilized states: From laboratory to large scale using a membrane contactor

Highlights

• Preparation of clove essential oil-in-cyclodextrin-in-liposomes (DCL) at laboratory and large scales.

• Freeze-drying of liposomes encapsulating inclusion complex of a volatile hydrophobic compound.

• DCL formulations were stable in aqueous dispersions after 1 month of storage at 4 ̊C and in lyophilized forms.

• DCL formulations maintained the DPPH scavenging activity of free clove essential oil, free eugenol and their inclusion complexes.

Abstract

This work is dedicated to prepare liposomal dry powder formulations of inclusion complexes of clove essential oil (CEO) and its main component eugenol (Eug). Ethanol injection method and membrane contactor were applied to prepare liposomes at laboratory and large scale, respectively. Various liposomal formulations were tested: (1) free hydroxypropyl-β-cyclodextrin loaded liposomes; (2) drug in hydroxypropyl-β-cyclodextrin in liposomes (DCL); (3) DCL₂ obtained by double loading technique, where the drug is added in the organic phase and the inclusion complex in the aqueous phase. Liposomes were characterized for their particle size, polydispersity index, Zeta potential, morphology, encapsulation efficiency of CEO components and Eug loading rate. Reproducible results were obtained with both injection devices. Compared to Eug-loaded liposomes, DCL and DCL₂ improved the loading rate of Eug and possessed smaller vesicles size. The DPPH^• scavenging activity of Eug and CEO was maintained upon incorporation of Eug and CEO into DCL and DCL₂. Contrary to DCL₂, DCL formulations were stable after 1 month of storage at 4 °C and upon reconstitution of the dried lyophilized cakes. Hence, DCL in aqueous and lyophilized forms, are considered as a promising carrier system to preserve volatile and hydrophobic drugs enlarging their application in cosmetic, pharmaceutical and food industries.

Introduction

As natural products, essential oils are gaining increasing interest in several areas. In the pharmaceutical field, they are included in the composition of many dosage forms (capsules, ointments, creams, syrups, suppositories, aerosols and sprays), and in food industries as food preservatives and in packaging (El-Asbahani et al., 2015). In this study, clove essential oil (CEO) and its main component eugenol (Eug) are chosen as models of essential oils. This choice is based on the simplicity of CEO composition since the three main components eugenol, eugenyl acetate and β-caryophyllene represent more than 99% of CEO constituents (Alma et al., 2007, Edris and Malone, 2012). Moreover, CEO components possess remarkable biological effects including antioxidant (Baskaran et al., 2010, Teixeira et al., 2013), anti-inflammatory (Bachiega, De Sousa, Bastos, & Sforcin, 2012), antibacterial (Teixeira et al., 2013), antifungal (Vazquez, Fente, Franco, Vazquez, & Cepeda, 2001), antiviral (Hussein et al., 2000), anti-acaricidal (Kim & Sharma, 2011), anticarciogenic (Kouidhi, Zmantar, & Bakhrouf, 2010), analgesic, anesthetic (Jadhav, Khandelwal, Ketkar, & Pisal, 2004), and neuroprotective (Müller, Pape, Speckmann, & Gorji, 2006), making them strong potential agents in cosmetic, pharmaceutical and food industries. Eug is considered non-mutagenic, non-carcinogenic, and recognized as safe by Food and Drug administration. Eug is widely used as flavoring agent in a variety of food products such as baked food, sweets, beverages, and frozen dairy products (Baskaran et al., 2010). Besides, Eug is a component of creams, soap, detergent, perfumes, and lotion in USA market (Opdyke, 1975) and in many deodorants on the European markets (Rastogi et al., 1998).

However, CEO components are volatile, photo-sensible and possess low aqueous solubility. For these reasons, different carrier systems including cyclodextrins, cyclodextrin grafted chitosan, micelles, liposomes, solid lipid nanoparticles, poly(dl-lactide-co-glycolide) nanoparticles, chitosan nanoparticles, nanostructured lipid carriers, polycaprolactone nanocapsules, whey protein isolate and maltodextrin nanocapules, maltodextrin and gum Arabic matrices were developed to extend their applications (Greige-Gerges & Sebaaly, 2015).

Liposomes are microscopic vesicles consisting of a central aqueous compartment surrounded by one or more concentric phospholipid bilayers. They can encapsulate hydrophilic substances in the interior aqueous compartment, lipophilic drugs within lipid bilayers and amphiphilic molecules at the lipid/water interface (Laouini et al., 2012a). Due to their biocompatibility, biodegradability, and low toxicity, potential applications of liposomes as pharmaceutical carriers for efficacy enhancement and toxicity reduction are well recognized (Lian & Ho, 2001). However, the incorporation of poorly water-soluble drugs in lipid bilayers is often limited, and the hydrophobic molecules can be rapidly released from lipid bilayers (Maestrelli, González-Rodríguez, Rabasco, & Mura, 2005). This may limit the potential application of liposomes as carriers for hydrophobic molecules.

Nowadays, cyclodextrins (CDs) are gaining increasing interest due to their ability to form inclusion complexes with a wide range of water insoluble or unstable molecules. Encapsulation with CDs can increase their aqueous solubility and improve their stability by protecting them from oxidation, thermal degradation, and evaporation (Marques, 2010). CDs are non-toxic cyclic oligosaccharides, consisting of six (α-CD), seven (β-CD) or eight (γ-CD) units of α (1–4) linked d-glucopyranose. They are obtained from enzymatic digestion of starch. CDs have an inner hydrophobic cavity formed by pyranose rings, and a hydrophilic external shell formed by primary and secondary hydroxyl groups from the glucose units (Yang et al., 2010). The CDs conformation allows the entrapment of hydrophobic molecules in the CDs internal cavity forming non-covalent inclusion complexes. 2-hydroxypropyl-β-CD (HP-β-CD), a CD derivative, is widely used on industrial scale since it has high aqueous solubility and a safe toxicity profile (Challa, Ahuja, Ali, & Khar, 2005). Hill, Gomes, and Taylor (2013) prepared β-CD inclusion complexes of various essential oils including Eug and CEO by freeze-drying method. The authors proved that β-CD increased the aqueous solubility of essential oils and protected them from oxidation. Moreover, β-CD/EO inclusion complexes inhibited Salmonella enterica serovar Typhimurium and Listeria innocua growth at lower active compound concentrations than free oils (Hill et al., 2013).

Supramolecular systems of polyvinyl alcohol nanofibers containing CD/Eug inclusion complex (Kayaci, Ertas, & Uyar, 2013) or swollen micelles (Kriegel, Kit, McClements, & Weiss, 2010) have been developed. These systems demonstrated high thermal stability and a controlled release of Eug (Kayaci et al., 2013, Kriegel et al., 2010). The concept of joining liposomes and cyclodextrin/drug complex forming drug in cyclodextrin in liposomes (DCL) formulations was proposed first by McCormack and Gregoriadis (1994), in order to avoid the drawbacks associated with liposomes or CDs in a single system and combine the advantages of both carriers. DCL would allow the accommodation of insoluble drugs in the aqueous phase of vesicles (Nasir, Harikumar, & Amanpreet, 2012), and supposed to be able to increase the stability and the encapsulation efficiency (EE) of volatile molecules to levels above those attained by drug incorporation in lipid bilayers. This study is dedicated to explore the ability of this combined system to improve the characteristics of lipid vesicles containing Eug or CEO (Sebaaly, Jraij, Fessi, Charcosset, & Greige-Gerges, 2015).

Membrane contactors, applied to the preparation of colloids such as nanoparticles, nanocapsules, solid lipid nanoparticles, nano-emulsions, and liposomes (Charcosset and Fessi, 2005a, Charcosset et al., 2005b, Jaafar-Maalej et al., 2010, Khayata et al., 2012, Laouini et al., 2012b) have also received much interest. The main advantages of membrane contactors for the preparation of colloids are their ability to control the colloids size by an appropriate choice of process parameters, and to increase the production rate by increasing the membrane area. In this study we applied for the first time the membrane contactor based on the principles of the ethanol injection method for DCL preparation.

On the other hand, liposomes can be stored as an aqueous dispersion or on a freeze-dried state (Crommelin & Van Bommel, 1984). Freeze-drying may increase the shelf life of liposomes. Since CDs are well known for their cryoprotectant ability during lyophilization (Van den Hoven, Metselaar, Storm, Beijnen, & Nuijen, 2012), in this study we will investigate whether DCL formulations are stable during freeze-drying. Producing a dry powder formulation of liposomes encapsulating a volatile and poorly water-soluble molecule is a key challenge, since the drug can leak out from the liposomes during drying. This approach has not been investigated yet to the best of our knowledge.

In the present study, DCL systems were developed at laboratory and large scale using a membrane contactor. Inclusion complexes of Eug and CEO were prepared and then encapsulated into liposomes. Freeze-drying was also applied for the formulations prepared at small scale. Blank, Eug- and CEO-loaded DCL systems were characterized for their size, polydispersity index (pdI), zeta potential, morphology, encapsulation efficiency, loading rate and stability and assessed for their 2,2-diphenyl-1-picrylhydrazil (DPPH•) scavenging activity.